Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) transmembrane subunit gp41 plays a crucial role in the early steps of viral entry into target cells (38) and may serve as an important target for development of HIV-1 entry inhibitors (4, 25). The gp41 molecule consists of three domains, i.e., cytoplasmic domain, transmembrane domain and extracellular domain (ectodomain). The ectodomain contains three major functional regions: the fusion peptide (FP), the N-terminal heptad repeat (NHR or HR1) and the C-terminal heptad repeat (CHR or HR2). Peptides derived from the NHR and CHR regions of gp41, designated N- and C-peptides, have potent antiviral activity against HIV-1 infection (21, 33, 49, 50). One of the C-peptides, T-20 (previously known as DP-178 and now as Fuzeon), has shown potent in vivo anti-HIV-1 activity in clinical trials for treatment of patients with HIV-1 infection and AIDS (27, 50) and was recently approved by the US FDA as the first member of a new class of anti-HIV drugs, known as HIV fusion inhibitors. Discovery of this drug is a great breakthrough in the development of anti-HIV drugs since it can be used for treatment of HIV-infected individuals who fail to respond to the currently available anti-retroviral drugs, such as HIV reverse transcriptase and protease inhibitors (26, 29). However, the future application of T-20 may be constrained due to its lack of oral availability and high cost of production. Therefore, it is essential to develop small molecule anti-HIV-1 compounds with a mechanism of action similar to that of C-peptides but without the disadvantages of the peptidic drugs.
In the study on the mechanism by which C-peptides inhibit HIV-1 fusion, it has been demonstrated that the gp41 N- and C-peptides mixed at equimolar concentrations form a stable α-helical trimer of antiparallel heterodimers, representing the fusion-active gp41 core (33, 35). Crystallographic analysis has revealed that this is a six-stranded α-helical bundle, in which three N-helices associate to form the central trimeric coiled-coil and three C-helices pack obliquely in an antiparallel manner into the highly conserved hydrophobic grooves on the surface of this coiled-coil (3, 46, 48). The C-helix interacts with the N-helix mainly through the hydrophobic residues in the grooves on the surface of the central coiled-coil trimer. Each of the grooves on the surface of the N-helix trimer has a deep hydrophobic pocket that accommodates three conserved hydrophobic residues in the gp41 CHR region (3), suggesting that this pocket is an attractive target for designing new class of anti-HIV-1 drugs, which may prevent the early fusion events (2, 3).
Using the gp41 pocket as the target structure, previously two small molecule compounds were identified, ADS-J1 (9, 19) and XTT formazan (51) by applying a computer-aided molecular docking techniques and a sandwich enzyme linked immunosorbent assay (ELISA) (23) using a monoclonal antibody (mAb), NC-1, which specifically recognizes the fusion-active gp41 core structure (20). These compounds inhibit HIV-1 fusion possibly by docking into the gp41 pocket and interfering with the formation of the gp41 six-helix bundle formation. However, they may not be good lead compounds for development of anti-HIV-1 drugs since both are dyes and contain several reactive groups. Nevertheless, the identification of these compounds is useful as a proof of concept that a small molecule organic compound might block the fusion-active gp41 six-helix bundle formation and inhibit HIV-1 entry. Here it is reported that the identification of two pyrrole derivatives, designated NB-2 and NB-64, as novel HIV-1 fusion inhibitors, which may interact with gp41 at the fusion-intermediate conformation, possibly binding to the gp41 hydrophobic pocket and surrounding area and block the gp41 six-helix bundle formation, thereby inhibiting the fusion between the viral and target cell membranes. NB-2 and NB-64 are “drug-like” compounds and may be used as leads for designing more potent HIV-1 entry inhibitors, which are expected to be developed as a new class of anti-HIV-1 drugs.